Recombinant follicle-stimulating hormone (FSH) (follitropin alfa) and biosimilar preparations are available for clinical use. They have specific FSH activity and a unique glycosylation profile dependent on source cells. The aim of the study is to compare the originator (reference) follitropin alfa (Gonal-f (R))- with biosimilar preparations (Bemfola (R) and Ovaleap (R))-induced cellular responses in vitro. Gonadotropin N-glycosylation profiles were analyzed by ELISA lectin assay, revealing preparation specific-patterns of glycan species (Kruskal-Wallis test; p < 0.05, n = 6) and by glycotope mapping. Increasing concentrations of Gonal-f (R) or biosimilar (1 x 10(-3) -1 x 10(3) ng/ml) were used for treating human primary granulosa lutein cells (hGLC) and FSH receptor (FSHR)-transfected HEK293 cells in vitro. Intracellular cAMP production, Ca2+ increase and beta-arrestin 2 recruitment were evaluated by BRET, CREB, and ERK1/2 phosphorylation by Western blotting. 12-h gene expression, and 8- and 24-h progesterone and estradiol synthesis were measured by real-time PCR and immunoassay, respectively. We found preparation-specific glycosylation patterns by lectin assay (Kruskal-Wallis test; p < 0.001; n = 6), and similar cAMP production and beta-arrestin 2 recruitment in FSHR-transfected HEK293 cells (cAMP EC50 range = 12 +/- 0.9-24 +/- 1.7 ng/ml; beta-arrestin 2 EC50 range = 140 +/- 14.1-313 +/- 18.7 ng/ml; Kruskal-Wallis test; p >= 0.05; n = 4). Kinetics analysis revealed that intracellular Ca2+ increased upon cell treatment by 4 mu g/ml Gonal-f (R), while equal concentrations of biosimilars failed to induced a response (Kruskal-Wallis test; p < 0.05; n = 3). All preparations induced both 8 and 24 h-progesterone and estradiol synthesis in hGLC, while no different EC(50)s were demonstrated (Kruskal -Wallis test; p > 0.05; n = 5). Apart from preparation-specific intracellular Ca2+ increases achieved at supra-physiological hormone doses, all compounds induced similar intracellular responses and steroidogenesis, reflecting similar bioactivity, and overall structural homogeneity.

Glycosylation Pattern and in vitro Bioactivity of Reference Follitropin alfa and Biosimilars / Riccetti, Laura; Sperduti, Samantha; Lazzaretti, Clara; Klett, Danièle; De Pascali, Francesco; Paradiso, Elia; Limoncella, Silvia; Potì, Francesco; Tagliavini, Simonetta; Trenti, Tommaso; Galano, Eugenio; Palmese, Angelo; Satwekar, Abhijeet; Daolio, Jessica; Nicoli, Alessia; Villani, Maria Teresa; Aguzzoli, Lorenzo; Reiter, Eric; Simoni, Manuela; Casarini, Livio. - In: FRONTIERS IN ENDOCRINOLOGY. - ISSN 1664-2392. - 10:(2019), pp. 1-13. [10.3389/fendo.2019.00503]

Glycosylation Pattern and in vitro Bioactivity of Reference Follitropin alfa and Biosimilars

Riccetti, Laura;Sperduti, Samantha;Lazzaretti, Clara;Paradiso, Elia;Daolio, Jessica;Reiter, Eric;Simoni, Manuela;Casarini, Livio
2019

Abstract

Recombinant follicle-stimulating hormone (FSH) (follitropin alfa) and biosimilar preparations are available for clinical use. They have specific FSH activity and a unique glycosylation profile dependent on source cells. The aim of the study is to compare the originator (reference) follitropin alfa (Gonal-f (R))- with biosimilar preparations (Bemfola (R) and Ovaleap (R))-induced cellular responses in vitro. Gonadotropin N-glycosylation profiles were analyzed by ELISA lectin assay, revealing preparation specific-patterns of glycan species (Kruskal-Wallis test; p < 0.05, n = 6) and by glycotope mapping. Increasing concentrations of Gonal-f (R) or biosimilar (1 x 10(-3) -1 x 10(3) ng/ml) were used for treating human primary granulosa lutein cells (hGLC) and FSH receptor (FSHR)-transfected HEK293 cells in vitro. Intracellular cAMP production, Ca2+ increase and beta-arrestin 2 recruitment were evaluated by BRET, CREB, and ERK1/2 phosphorylation by Western blotting. 12-h gene expression, and 8- and 24-h progesterone and estradiol synthesis were measured by real-time PCR and immunoassay, respectively. We found preparation-specific glycosylation patterns by lectin assay (Kruskal-Wallis test; p < 0.001; n = 6), and similar cAMP production and beta-arrestin 2 recruitment in FSHR-transfected HEK293 cells (cAMP EC50 range = 12 +/- 0.9-24 +/- 1.7 ng/ml; beta-arrestin 2 EC50 range = 140 +/- 14.1-313 +/- 18.7 ng/ml; Kruskal-Wallis test; p >= 0.05; n = 4). Kinetics analysis revealed that intracellular Ca2+ increased upon cell treatment by 4 mu g/ml Gonal-f (R), while equal concentrations of biosimilars failed to induced a response (Kruskal-Wallis test; p < 0.05; n = 3). All preparations induced both 8 and 24 h-progesterone and estradiol synthesis in hGLC, while no different EC(50)s were demonstrated (Kruskal -Wallis test; p > 0.05; n = 5). Apart from preparation-specific intracellular Ca2+ increases achieved at supra-physiological hormone doses, all compounds induced similar intracellular responses and steroidogenesis, reflecting similar bioactivity, and overall structural homogeneity.
10
1
13
Glycosylation Pattern and in vitro Bioactivity of Reference Follitropin alfa and Biosimilars / Riccetti, Laura; Sperduti, Samantha; Lazzaretti, Clara; Klett, Danièle; De Pascali, Francesco; Paradiso, Elia; Limoncella, Silvia; Potì, Francesco; Tagliavini, Simonetta; Trenti, Tommaso; Galano, Eugenio; Palmese, Angelo; Satwekar, Abhijeet; Daolio, Jessica; Nicoli, Alessia; Villani, Maria Teresa; Aguzzoli, Lorenzo; Reiter, Eric; Simoni, Manuela; Casarini, Livio. - In: FRONTIERS IN ENDOCRINOLOGY. - ISSN 1664-2392. - 10:(2019), pp. 1-13. [10.3389/fendo.2019.00503]
Riccetti, Laura; Sperduti, Samantha; Lazzaretti, Clara; Klett, Danièle; De Pascali, Francesco; Paradiso, Elia; Limoncella, Silvia; Potì, Francesco; Tagliavini, Simonetta; Trenti, Tommaso; Galano, Eugenio; Palmese, Angelo; Satwekar, Abhijeet; Daolio, Jessica; Nicoli, Alessia; Villani, Maria Teresa; Aguzzoli, Lorenzo; Reiter, Eric; Simoni, Manuela; Casarini, Livio
File in questo prodotto:
File Dimensione Formato  
2019 Riccetti et al - Frontiers in Endocrinology.pdf

accesso aperto

Tipologia: Versione dell'editore (versione pubblicata)
Dimensione 2.67 MB
Formato Adobe PDF
2.67 MB Adobe PDF Visualizza/Apri
Pubblicazioni consigliate

Caricamento pubblicazioni consigliate

Licenza Creative Commons
I metadati presenti in IRIS UNIMORE sono rilasciati con licenza Creative Commons CC0 1.0 Universal, mentre i file delle pubblicazioni sono rilasciati con licenza Attribuzione 4.0 Internazionale (CC BY 4.0), salvo diversa indicazione.
In caso di violazione di copyright, contattare Supporto Iris

Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11380/1181078
Citazioni
  • ???jsp.display-item.citation.pmc??? 10
  • Scopus 13
  • ???jsp.display-item.citation.isi??? 14
social impact